Prostate cancer is the most frequently diagnosed male cancer and second leading cause of male cancer death. If primary treatment modalities are not capable of controlling the disease, inhibiting androgen receptor (AR) activity is an effective treatment for advanced prostate cancer patients. However, the duration of patient responses to AR-targeted therapy is variable, and all patients will ultimately develop resistance and progress to a castration-resistant form of the disease associated with morbidity and death. One important feature of castration-resistant prostate cancer (CRPC) is aberrant re-activation of AR transcriptional activity despite reduced levels of circulating androgens. This knowledge has led to the development of new therapies that inhibit mechanisms supporting residual transcriptional activity of the AR in CRPC. However, resistance remains a major limitation for these new AR-targeted agents. Because of the major need for new understanding and innovation in this area, the long-term objectives of this research are to determine the mechanisms that can support persistent AR transcriptional activity despite AR-targeted therapy, and to develop more effective strategies for treating and managing patients with CRPC. The central discovery driving the proposed studies is altered AR mRNA splicing, giving rise to COOH-terminally truncated AR variant (AR-V) proteins that achieve constitutive transcriptional activity in the complete absence of androgen stimulus. Synthesis of these AR-Vs has emerged as an important aspect of clinical CRPC progression, but mechanisms responsible for alterations in AR splicing are largely unknown. The overarching goal of this proposal is to understand the role of newly-discovered AR gene rearrangements in driving altered AR splicing patterns and efficient AR-V synthesis in CRPC. To achieve this goal, three specific aims are proposed. In Aim 1, an arsenal of customized next-generation DNA and RNA sequencing tools will be used to determine the spectrum of AR gene rearrangements and splicing alterations in tissues from patients with CRPC. In Aim 2, programmable nuclease technology will be used to experimentally engineer site-specific AR gene rearrangements in the prostate cancer genome. These genome-engineered cells will be studied to establish the links between architectural changes in the AR gene, aberrant AR mRNA splicing patterns, AR-V synthesis, and the CRPC phenotype. In Aim 3, AR gene rearrangements will be evaluated as new biomarkers for detecting AR-V driven CRPC cell populations in heterogeneous tumors, predicting responses to AR-targeted therapy, and monitoring the development of resistance during AR-targeted therapy. Success with these studies will establish the role of an entirely new class of AR gene alterations in prostate cancer progression, and provide new methods for evaluating AR gene status in prostate cancer cells. Knowledge and resources developed in this proposal will be important for developing new treatment regimens for prostate cancer based on an individual patient's unique AR gene architecture.